s2fieldsofstudy: Biology Engineering

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Cell Factory Engineering of Undomesticated Bacillus Strains Using a Modified Integrative and Conjugative Element for Efficient Plasmid Delivery

A large number of Bacillus strains have been isolated from various environments and many of them have great potential as cell factories. However, they have been rarely developed as cell factories due to their poor transformation efficiency. In this study, we developed a highly efficient plasmid delivery system for undomesticated Bacillus strains using a modified integrative and conjugative element (MICE), which was designed to be activated by an inducer, prevent self-transfer, and deliver desired plasmids to the recipient cells. The MICE system was demonstrated to successfully introduce a gfp-containing plasmid into all 41 undomesticated Bacillus subtilis strains tested and eight other Bacillus species. The MICE was used to deliver a cytosine base editor (CBE)-based multiplex genome-editing tool for the cell factory engineering of the Bacillus species. The introduced CBE enabled one-step inactivation of the major extracellular protease genes of the tested strains. The engineered strains were used as hosts for heterologous expression of nattokinase, which resulted in various enzyme expression levels. The results suggested that the MICE and CBE systems can be powerful tools for genetic engineering of undomesticated Bacillus strains, and greatly contribute to the expansion of the Bacillus cell factory.

INTRODUCTION
Bacillus species are among the most important industrial microorganisms that have been widely used for a variety of applications like the production of various enzymes and metabolites, as agricultural agents, in traditional food fermentations, and as hosts for heterologous protein expression (Caulier et al., 2019;Su et al., 2020). The advantages of Bacillus as a protein-production host are that it can secrete large amounts of protein from the cells, mass culture is well established, and most of the Bacillus strains are safe for humans (Westers et al., 2004). There are recent reports showing the potential of Bacillus strains for medical use. Fengycin, produced by Bacillus species eliminates the pathogen Staphylococcus aureus from the human body (Piewngam et al., 2018). Many health benefits as probiotics of Bacillus species such as antioxidant, anti-cancer, anti-diabetic and immuno-modulatory effects have been documented (Elshaghabee et al., 2017).
The genus Bacillus consists of more than 280 species (Gupta et al., 2020). The domesticated Bacillus subtilis strain 168 has been largely handled as a cell factory because its physiology and genetics are extensively studied. Reportedly, undomesticated ATCC 6051 strain has a greater potential as alternative industrial host compared to strain 168, as it doesn't exhibit auxotrophy and can reach higher cell densities on complex media (Kabisch et al., 2013). Studies using not only B. subtilis but also Bacillus licheniformis (Song et al., 2021), Bacillus megaterium (Vary et al., 2007), and Bacillus pumilus (Küppers et al., 2014) as industrial protein production hosts have been reported. The promising properties related to the production of specific enzymes and other secondary metabolites have been documented in less-characterized strains such as Bacillus sonorensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus vallismortis, and Bacillus mojavensis (Heinze et al., 2018). In addition, certain Bacillus strains exhibit characteristics such as high resistance to toxic substrates or organic solvents (Kataoka et al., 2011;Mahipant et al., 2019), or can grow over extended ranges of salinity (Ibarra-Villarreal et al., 2021), pH (Newcombe et al., 2009) or temperature (Choonut et al., 2020), highlighting the potential of undomesticated Bacillus strains.
Undomesticated strains often need to be engineered to meet industrial demands. But, poor transformation efficiency has greatly limited their engineering. Transformation of Bacillus strains is usually performed through natural competence, electroporation, or conjugation. Natural competence method is commonly used in B. subtilis and depends on the expression of the competence machinery genes (Dubnau and Blokesch, 2019). Electroporation is also frequently used in various Bacillus strains (Zhang et al., 2015). Transformation of undomesticated Bacillus strains using these two methods is frequently inefficient or even impossible, while conjugation methods often solve the problem due to their broad-host-range nature. The well-known conjugal DNA transfer of Bacillus is assisted by a complex machinery for DNA translocation encoded in the Escherichia coli S17-1 chromosome (Simon et al., 1983) or B. subtilis natto plasmid pLS20 (Koehler and Thorne, 1987). However, many undomesticated strains are difficult to transform even with conjugation. Recently, it was reported that DNA was efficiently transferred to various undomesticated bacteria using engineered chromosomal integrative and conjugative elements (ICE) of B. subtilis (Brophy et al., 2018). However, the engineered system is difficult to use for marker-free genome engineering or serial editing of multiple genes in recipient cells, because it integrates the transferred DNAs with a selectable marker into a specific site of the recipient genome. Therefore, an effective plasmid delivery system is required in the marker-free engineering of undomesticated Bacillus strains for cell factory development.
Bacillus genome engineering for industrial applications has been carried out in a marker-free manner via counter-selectable markers, such as upp (Fabret et al., 2002), araR (Liu et al., 2008), mazF (Zhang et al., 2006), and a synthetic gene circuit (Jeong D. E. et al., 2015). However, recent editing tools are focused on a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 and its derivatives. The sequence-specific target programming capacity of Cas9 has made it a powerful and indispensable genome-editing tool (Tyagi et al., 2020). In B. subtilis, several highly efficient genome-editing protocols using the CRISPR-Cas9 (Altenbuchner, 2016;Westbrook et al., 2016;So et al., 2017), and a subsequent serial genome-editing method for multiple targets have been reported (Lim and Choi, 2019). Other systems, such as CRISPR/Cpf1 assisted multiple-genes editing (Wu et al., 2020), CRISPRi-guided multiplexed fine-tuning of metabolic flux (Dong et al., 2020), and CRISPR/Cas9n-based multiplex genome editing (Liu et al., 2019) have also been documented. In addition, a tool called cytosine base editor (CBE), in which a nuclease-deactivated Cas9 (dCas9) is fused with a cytidine deaminase, has been developed for multiplex genome editing in B. subtilis (Kim et al., 2021). Most CRISPR-based genome-editing tools can be applied to undomesticated strains if an efficient plasmid delivery system is established.
In this study, an efficient plasmid transfer system was developed using a modified integrative and conjugative element (MICE) and applied to deliver a CBE-based multiplex genome editing tool for cell factory engineering of undomesticated Bacillus strains. We believe that the MICE system may be a powerful tool for the development of undomesticated Bacillus strains as cell factories.

Donor Construction
The plasmids and primers used in this study are listed in Supplementary Tables 3, 4, respectively. All-in-one CRISPR plasmid (pACas9T) used for donor construction was constructed as follows. To construct a pHPCas9spac, BamHI-and SmaIdigested pHPspac (Kim et al., 2014) was ligated with the BamHI-and SmaI-digested fragment amplified by PCR from pHCas9 (So et al., 2017) using primers Cas9F and Cas9R. Then, the rep-cat cassette and oriT e fragment [oriT for E. coli-to-Bacillus conjugation (EBC)] were PCR amplified using primers pADallF/pADallR and oriTF/oriTR from pAD123 (Dunn and Handelsman, 1999) and pHCas9, respectively, and were fused with XhoI-and NarI-digested pHCas9spac using the Cold Fusion Cloning Kit (System Biosciences Inc., Palo Alto, CA, United States). The resulting plasmid was digested with XhoI and BsrGI, and then fused with the sgRNA cassette amplified from pAgR (So et al., 2017) using primers gRNAF and gRNAR to construct pACas9T plasmid. The oligonucleotides containing the 20-bp gRNA generated by mixing synthetic primers (Supplementary Table 4) were ligated with the AarI -digested pACas9T plasmid. The sgRNA synthesis was under the control of P ara promoter (So et al., 2017). For cloning of the donor DNA cassette amplified by fusion PCR using primers listed in Supplementary Table 4, pACas9T containing targeting gRNA was digested with SpeI and XmaI, and the large fragment was fused to the donor DNA cassette using a cold fusion cloning kit to produce the following CRISPR plasmids: pACas9T-dalrA, pACas9T-dnicK, and pACas9T-dsigK (Supplementary Table 3). For CRISPR-Cas9 editing, the CRISPR plasmid-containing cells were spread on an LB agar plate supplemented with 1 mM IPTG and chloramphenicol (5 µg/ml). Resulting colonies were screened by colony PCR to confirm the mutation. The plasmid curing was performed as described previously (So et al., 2017).
To construct the BBC donor, B. subtilis BS5418 (B. subtilis BSK1 thrC::P xyl -comK) was constructed by transferring the BS5417 chromosome containing thrC::P xyl -comK (Jeong D. E. et al., 2015) to B. subtilis BSK1 (Choi et al., 2009). The transfer of plasmid pLS20 from B. subtilis natto KCCM 12512 to BS5418 was performed as follows: a plasmid pBC16 (Palva et al., 1990) was introduced into B. subtilis natto KCCM 12512 by electroporation as previously described (Meddeb-Mouelhi et al., 2012) to construct a strain BS5879 containing both pLS20 and pBC16. We used strain BS5879 as a donor cell for co-transferring the plasmids pBC16 and pLS20 to the recipient strain BS5418. The pLS20-mediated conjugation was performed as described in the section "Transformation" below. The introduction of both pLS20 and pBC16 into BS5418 was confirmed by colony PCR. To retain only pLS20 in BS5418, plasmid pBC16 was eliminated by introducing pACas9T-gTc with tet gene-targeting gRNA. After confirming the removal of pBC16 by colony PCR, curing of pACas9T-gTc was performed to obtain the strain BS5887. A previous report showed that deletion of alanine racemase-encoding gene alrA in B. subtilis resulted in D-alanine auxotrophy that could be used as a counter-selectable marker against donor growth (Brophy et al., 2018). To construct the alrA deletion mutant, pACas9T-dalrA was introduced into BS5887, and the resulting colonies were screened by colony PCR to confirm the deletion of the alrA gene. After curing the plasmid pACas9T-dalrA, the resulting strain BS5892 was used as a donor strain in the BBC experiment.
Modified integrative and conjugative element donor contain deletion from rapI to attR, insertion of rapI gene into alrA gene, and mutation of oriT in the nicK gene. For the deletion from rapI to attR, two DNA fragments corresponding to upstream and downstream regions of rapI-attR were amplified from the chromosome of B. subtilis 168 using primers yddJ-H3F/yddJ-hygR and yddN-hygF/yddN-EcoR, respectively. The hygromycin-resistance gene (hygR) was amplified from the chromosome of B. subtilis WB800N  using primers hyg-F and hyg-R, and fused to the upstream and downstream fragments of rapI-attR by fusion PCR to obtain rapI::hygR cassette. Plasmid pUC18 was digested with HindIII and EcoRI, and the large fragment was fused to the rapI::hygR cassette using a cold fusion cloning kit to construct plasmid pUC-rapI::hyg. The resulting plasmid was introduced into B. subtilis 168 to generate strain BS5889. Next, pGEM-rapI was constructed as follows to generate a mutation in alrA gene for inducible rapI expression. Three DNA fragments corresponding to upstream and downstream regions of alrA, and structural gene of rapI were amplified from the chromosome of B. subtilis 168 using primers alr_front_F/alr_front_R, alr_back_F/alr_back_R, and rapI-F/rapI-R, respectively. The spectinomycin-resistance gene (spcR) and inducible promoter P xylA was amplified from the pDG1728 (Guérout-Fleury et al., 1996) and pAX01 (Härtl et al., 2001) using spcF/spcR and xyl-F/xyl-R, respectively. All fragments were fused by fusion PCR to obtain alrA::spcR-P xyl -rapI cassette. pGEM-T Easy (Promega Co., Madison, WI, United States) was digested with NcoI and PstI, and the large fragment was fused to the alrA::spcR-P xyl -rapI cassette using a cold fusion cloning kit to construct plasmid pGEM-rapI. The resulting plasmid was introduced into BS5889 to generate strain BS5890. Finally, to eliminate the oriT ICE (ICEBs1 origin of transfer) nicking site located within the nicK open reading frame (ORF), we changed nucleotide sequences of the oriT ICE without changing the amino acid sequence of the NicK. Plasmid pACas9T-dnicK for oriT ICE mutation was introduced into the BS5890. After confirming the oriT ICE mutation by sequencing and subsequent curing of the plasmid pACas9T-dnicK, the resulting strain BS5899 was used as a major donor strain in MICE experiments. MICE donor was further mutated in the sigK gene by introducing plasmid pACas9T-dsigK into BS5899. The resulting strain BS5918 is a sporulation-negative mutant, which can help the screening of transconjugants during conjugation.

Construction of Mobilizable Plasmids
The mobilizable plasmids pA3D-gfpTe, pA3D-gfpT20, and pA3D-gfpTi for expressing GFP under the control of cry3Aa promoter were constructed as follows. The cry3Aa promoter was amplified from the plasmid pMar3g  using primers 3Aag-F2 and stabR2. The gfp gene was amplified from plasmid pAD123 using primers gfp-F1 and gfp-R1, and fused to the cry3Aa promoter by fusion PCR to obtain the P cry3Aa -gfp cassette. The PCR product containing the P cry3Aagfp cassette was digested with EcoRI and BsiWI and inserted into corresponding sites of plasmid pDG1661 (Guérout-Fleury et al., 1996) to construct the pD3D-gfp. The plasmid pD3Dgfp was digested with EcoRI and NsiI, and the small fragment was ligated with EcoRI-and NsiI-digested plasmid pAD123 to construct a pA3D-gfp. For EBC, the oriT e region was amplified from plasmid pAgR using primers oriTe-AatF and oriTe-SacR. Plasmid pA3D-gfp was digested with AatII and SacI, and the large fragment was fused to the oriT e using a cold fusion cloning kit to construct pA3D-gfpTe. For BBC, the oriT 20 (pLS20 oriT) region was amplified by colony PCR using primers oriT20-BamF and oriT20-XbaR from B. subtilis natto KCCM 12512. Plasmid pA3D-gfpTe was digested with BamHI and XbaI, and the large fragment was fused to the oriT 20 using a cold fusion cloning kit to construct pA3D-gfpT20. For MICE, the oriT ICE (ICEBs1 oriT) region was amplified by PCR using primers oriTICE-BamF and oriTICE-XbaR from the chromosome of B. subtilis 168. Plasmid pA3D-gfpTe was digested with BamHI and XbaI, and the large fragment was fused to the oriT ICE using a cold fusion cloning kit to construct pA3D-gfpTi. The mobilizable plasmid pm3D-aprNTi for the expression of a nattokinase gene aprN under the control of cry3Aa promoter was constructed as follows. The plasmid pA3D-gfpTi was digested with AatII and NsiI, and the small fragment was ligated with AatII-and NsiI-digested plasmid pMGoldi-sCBE4 to construct pm3D-gfpTi. The cry3Aa promoter was obtained by PCR from pA3D-gfpTi using primers P3D-sacF and stabR2. The aprN structural gene was amplified from the chromosome of B. subtilis natto KCCM 12027 using primers aprE-F2 and aprE-BWR, and fused to the cry3Aa promoter by fusion PCR to obtain the P cry3Aa -aprN cassette. Plasmid pm3D-gfpTi was digested with SacI and BsiWI, and the large fragment was fused to the P cry3Aa -aprN cassette using a cold fusion cloning kit to construct pm3D-aprNTi.

Transformation
Transformation of B. subtilis through natural competence (Lim and Choi, 2019) and electroporation (Meddeb-Mouelhi et al., 2012) were carried out by previously described methods. EBC was performed as described previously with the following modifications (Teng et al., 1998). The E. coli S17-1 donor and B. subtilis recipient cells were grown at 37 • C for 16 h in LB broth with appropriate antibiotics. The cultures were diluted 100-fold in fresh LB broth and further incubated until OD 600 reached 0.6-0.8. Then, 1 ml of donor and recipient cells were harvested by centrifugation (10,000 × g, 3 min), washed once in 1 ml phosphate buffered saline (PBS, pH 7.2, Enzynomics, Daejeon, South Korea), and resuspended in 20 µl of LB broth. Then, 20 µl each of donor and recipient cell suspensions were mixed and spotted onto nitrocellulose membrane (0.45 µm, Advantec, Tokyo, Japan) placed on LB agar. After mating for 24 h at 37 • C, the filter was transferred to a 5-ml centrifuge tube containing 1 ml of LB broth and vortexed for 1 min to remove the bacteria from the filter. The resulting suspension was serially diluted and plated on LB agar containing polymyxin B and chloramphenicol for selection of transconjugants. All the plates were incubated for 24 h at 37 • C. BBC was performed as described previously with the following modifications (Koehler and Thorne, 1987). Donor and recipient strains were grown for 16 h in appropriate media and cultivation conditions (Supplementary Table 1). The cultures were diluted 20-fold with the same medium and cultured for 5 h. Then, 0.1 ml each of donor and recipient cell cultures were mixed, and 50 µl of the mixtures were spotted onto nitrocellulose membrane placed on LB agar containing 100 µg/ml D-alanine. After mating for 16 h at 37 • C, the filter was transferred to a 5ml centrifuge tube containing 1 ml PBS and vortexed for 1 min to remove the bacteria from the filter. The resulting suspension was serially diluted and plated on LB agar plate containing appropriate antibiotics for selection of transconjugants. All the plates were incubated for 24 h at 37 • C. MICE-based conjugation was performed as described previously with the following modifications (Brophy et al., 2018). Donor and recipient strains were grown for 16 h in appropriate media and cultivation conditions (Supplementary Table 1). The cultures were diluted 100-fold with the same medium and further cultured until OD 600 reached 1. During growth, expression of rapI was induced by adding 1% xylose to the cultures at OD 600 = 0.2. After mixing 3 ml each of donor and recipient cultures, the mixture was compressed using a syringe onto a 0.45-µm nitrocellulose filter and the filter was placed on LB agar or TSA (tryptic soy agar, Difco, Detroit, MI, United States) containing 100 µg/ml D-alanine. After mating for 3 h (5 h for B. megaterium and 24 h for B. vallismortis) at 30 • C or 37 • C (refer to Supplementary Table 1), the filter was transferred to a 5-ml centrifuge tube containing 1 ml PBS and vortexed for 1 min to remove the bacteria from the filter. The resulting suspension was serially diluted and plated on LB agar or TSA plate containing appropriate antibiotics for selection of transconjugants. All the plates were incubated for 24 h at 30 • C or 37 • C. When the sigK mutant BS5918 was used as the donor strain, the mating filters were placed on LB agar containing D-alanine and incubated for 7 days at 30 • C to ensure complete sporulation of the recipient. After suspending the filter with PBS, the suspension was treated at 80 • C for 60 min to remove donor cells and plated on LB agar containing appropriate antibiotics for selection of transconjugants.

Cytosine Base Editor-Mediated Multiplex Genome Editing
For CBE mutagenesis, we replaced oriT e of pMGold-sCBE4 (Kim et al., 2021) with oriT ICE . The oriT ICE was amplified by PCR using primers ICE-oriT-F and ICE-oriT-R from the chromosome of B. subtilis 168. Plasmid pMGold-sCBE4 was digested with HindIII and SacI, and the large fragment was fused to the oriT ICE using a cold fusion cloning kit to construct pMGoldi-sCBE4. To construct pMGoldi-xCBE4 expressing CBE under the P xylA promoter, P xylA from B. megaterium was amplified from pAX01 using primers xylR-xyl-F and xylR-xyl-R. Plasmid pMGoldi-sCBE4 was digested with SacI and FseI, and the large fragment was fused to the P xylA using a cold fusion cloning kit to construct pMGoldi-xCBE4. The gRNAs used for multiplex genome editing (Supplementary Table 4) were designed and cloned using Golden-Gate assembly as previously reported (Kim et al., 2021). The CBE was under the inducible promoters P spac or P xylA , but a leaky expression without IPTG or xylose inducers was sufficient to induce the mutations. Transconjugants were analyzed by DNA sequencing to confirm the mutations. The plasmid curing of the identified mutants were performed and then used in further experiments.

Analytical Methods
For the measurement of GFP fluorescence, cells were grown for 16 h in tryptic soy broth (TSB; Difco, Detroit, MI, United States) medium. The culture was diluted 100-fold in fresh TSB medium Frontiers in Microbiology | www.frontiersin.org FIGURE 1 | Comparison of conventional transformation and conjugation methods for undomesticated Bacillus subtilis strains. Transformation efficiency for natural competence and electroporation was calculated as colony forming unit per µg DNA used. E. coli-to-Bacillus conjugation (EBC) used E. coli S17-1 as a donor. Bacillus-to-Bacillus conjugation (BBC) used B. subtilis 168 containing plasmid pLS20 as a donor. The conjugation efficiency for EBC and BBC were calculated by dividing the number of transconjugants by the number of recipient cells. The numbers are KCTC strain numbers and the 168 indicates B. subtilis strain 168. Three independent experiments were performed and the results are presented as closed circles. and cultured until they reached late stationary phase (18 h after entering stationary phase). The GFP fluorescence of the cultured samples were measured with a TriStar2 microplate reader (Berthold Technologies, Bad Wildbad, Germany) as previously reported . To determine protease activity, the supernatants of cultured samples were measured using azocasein as a substrate according to a previously described method . All measurements were carried out at least three times. All data generated or analyzed in this study are included in the published article.

Transformation of Undomesticated B. subtilis Strains
Engineering of undomesticated Bacillus strains is challenging due to poor transformation efficiency. Here, we compared several previously known transformation and conjugation methods on eight undomesticated B. subtilis strains obtained from Korean Collection for Type Cultures (KCTC) (Figure 1 and Supplementary Table 1). The results showed that the natural competence method transformed only the domesticated strain 168. Electroporation transformed four strains, 1021, 1023, 1028, and 1102. The 1021 strain was transformed twice in three independent trials, while the strains 1023 and 1102 were transformed only in one out of three. The other four strains were untransformed by both methods. Because conjugation sometimes solves the problem, it was introduced for transformation of undomesticated strains. In the EBC method, E. coli S17-1 strain carrying the transfer genes of IncP-type plasmid RP4 in the chromosomes was used as a donor (Simon et al., 1983). The EBC method was able to transform only the same four strains as electroporation. BBC used a B. subtilis 168 containing a plasmid pLS20 as a donor. The pLS20, a 65-kb-conjugative plasmid derived from B. subtilis natto, is capable of transferring plasmids containing a mobile origin (oriT) to recipient cells (Koehler and Thorne, 1987). The BBC method is more efficient than the other methods; but, strains 1021 and 1104 were still untransformed. Therefore, a broad-host-range method for difficult-to-transform strains is needed.

Modification of B. subtilis Integrative and Conjugative Element
Integrative and conjugative elements is a modular mobile genetic element located on the host chromosome and is an important tool of horizontal gene transfer (Figure 2A; Auchtung et al., 2016). The expression of ICE genes induces production of the type IV secretion machinery for conjugation, excision of the ICE from the chromosome, and transfer of the excised ICE to the recipient cells. In the recipient cell, the transferred singlestranded ICE undergoes circularization, second-strand synthesis, and integration into the genome (Figure 2B). Here, we modified the ICEBs1 to deliver desired plasmids into undomesticated Bacillus strains. The MICE comprises (1) deletion of oriT from the nicK gene, (2) deletion from rapI to attR, and (3) insertion of B. megaterium xylA promoter-regulated rapI gene into alrA gene (Figure 2A and Supplementary Table 2). In the ICEBs1, the NicK DNA relaxase binds to the oriT of the excised circular ICEBs1 and makes a nick for rolling circle replication (Johnson and Grossman, 2015). In the MICE system, the chromosomal oriT was removed to prevent relaxase-mediated nicking of the chromosome. This enables MICE to inhibit self-transfer and assist only target plasmid transfer. As the oriT is located in the nicK gene, the deletion of oriT was accomplished using the CRISPR-Cas9 system by mutation of the oriT sequence without changing amino acid sequence of the NicK. The attR deletion in MICE inhibits its excision from the chromosome. Expression of ICEBs1 genes for conjugation is repressed by the ImmR regulator, ImmR is regulated by the protease ImmA, which in turn is activated by RapI. At low cell densities, the AbrB transition state regulator binds to the rapI promoter and represses its transcription, eventually inhibiting the expression of ICEBs1 genes. High cell density at the stationary phase inhibits abrB expression, which induces rapI expression. The expressed RapI activates ImmA, which cleaves the ImmR repressor, thereby inducing the expression of ICEBs1 genes. When the secreted PhrI accumulates in the medium, it re-enters the cell and inhibits RapI activity (Auchtung et al., 2016). Thus, removal of the rapI and phrI from the chromosome in the MICE system separates the expression of the MICE genes from the regulatory pathway of the host cell. The rapI gene was cloned under the inducible promoter and inserted into the chromosome to enable induction of the MICE system when desired, following the strategy reported earlier (Brophy et al., 2018). The MICE system was used to transfer plasmid pA3D-gfpTi into the eight undomesticated B. subtilis strains. The result showed that MICE was able to transfer the plasmid into all eight strains ( Figure 2C).
Next, we applied the MICE system to transfer the gfpcarrying plasmid pA3D-gfpTi into 41 undomesticated B. subtilis strains listed in Supplementary Table 1, and confirmed that the MICE could transfer the plasmid into all the listed strains. Interestingly, GFP fluorescence intensity varied from strain to strain, indicating that the expression level of heterologous proteins may differ even within the same species (Figure 3).  Table 1). Transformation of these strains using the MICE system revealed that the plasmid pA3D-gfpTi was successfully introduced into all of them. Like undomesticated B. subtilis, the transformed QPS strains displayed varying expression levels of GFP (Figure 4).

Cytosine Base Editor-Mediated Multiplex Genome Engineering of Undomesticated Bacillus Strains
For the engineering of the QPS strains, we chose AprE-, NprE-, and WprA-like extracellular proteases as engineering targets as they can break down secreted heterologous proteins or mask the activity of heterologous proteases (Figure 5A). B. lentus showed weak extracellular protease activity on a TSA plate containing 1% skim milk (Figure 5B), but the corresponding gene could not be found in the genome sequence deposited in the National Center for Biotechnology Information (NCBI) database. B. licheniformis used in this study has neither a genome sequence in the NCBI database nor extracellular protease activity ( Figure 5B). Thus, except B. lentus and B. licheniformis, the other seven strains were engineered to inactivate the target extracellular protease genes. For multiplex genome engineering of these strains, we used a CBE system consisting of a dCas9, a Petromyzon marinus cytidine deaminase (PmCDA1), and a uracil DNA glycosylase inhibitor (UGI) (Figure 5C), which can generate stop codons within the multiple target genes simultaneously with high efficiency (Kim et al., 2021). The gRNAs designed to create a stop codon within each protease gene are presented in Supplementary Figure 1. The CBE plasmids containing multiple gRNAs targeting major extracellular proteases were constructed (Supplementary Table 3) and introduced into the QPS strains using the MICE system. Except B. vallismortis, the plasmids were successfully transferred to all QPS Bacillus strains tested. Four transconjugants of each QPS strains, except B. megaterium, were randomly selected, and the introduction of stop codons into target genes was analyzed by sequencing. We confirmed that all target genes were edited simultaneously with 100% efficiency. For B. megaterium, only two transconjugants were obtained. Sequencing analysis revealed that all three target genes were edited in one transconjugant. In the other transconjugant, two out of three target genes were edited. Transconjugants for B. vallismortis were not obtained through the MICE system. We assumed that the P spac promoter used for the CBE expression was not tightly controlled, and the leaky expression of CBE could have a toxic effect on the growth of B. vallismortis cells. Therefore, the promoter P spac was replaced by P xylA , which is known as a more tightly controlled promoter than P spac in B. subtilis (Bhavsar et al., 2001). In addition, the MICE donor was further mutated in the sigK gene to construct a sporulation-negative donor BS5918, which can aid in the screening of transconjugants. Using the new plasmid and donor, three B. vallismortis transconjugants containing the CBE plasmid were obtained. Sequencing analysis confirmed that all target genes were edited in the three transconjugants. The edited QPS strains showed no or significantly decreased protease activities on TSA plate containing 1% skim milk compared to wild-type (WT) strains ( Figure 5B). We reconfirmed the decreased protease activities of the edited QPS strains through the protease assay using azocasein as a substrate (Supplementary Figure 2).

Heterologous Expression of a Nattokinase in Engineered Qualified Presumption of Safety Strains
The engineered QPS strains were used as heterologous expression hosts for the expression of aprN encoding the fibrinolytic enzyme nattokinase. The plasmid pm3D-aprNTi for expressing aprN was constructed (Supplementary Table 3) and introduced into engineered QPS Bacillus strains and wild type of B. lentus and B. licheniformis through the MICE system. The results showed that all QPS strains were successfully transformed with the plasmid, and produced nattokinase. The protease activity of the nattokinase varied among QPS strains, and high activity was observed in B. amyloliquefaciens, B. subtilis, and B. mojavensis (Figure 6A). Nattokinase productivity in the QPS strains was confirmed through SDS-PAGE analysis ( Figure 6B). Taken together, we showed through this study that various undomesticated Bacillus strains can be engineered and used as cell factories.

DISCUSSION
The MICE system for highly efficient plasmid delivery into undomesticated Bacillus strains was modified from the previously  reported mini-ICEBs1 system (Brophy et al., 2018) since the previous system cannot be used for marker-free genome engineering of undomesticated Bacillus strains. Although the mini-ICEBs1 system exhibits broad-host-range characteristics, the ICEBs1 inhibits host cell acquisition of a second copy, which may reduce the efficiency of conjugation between the ICEBs1-harboring strains. To date, three inhibition mechanisms have been reported; inhibition of PhrI-mediated cell signaling, ImmR repressor-mediated immunity, and exclusion mechanism based on specificity between donor's conjugation protein ConG and recipient's exclusion protein YddJ (Avello et al., 2019). Both mini-ICEBs1 and the MICE systems circumvent the first mechanism by overexpressing the regulator RapI. The ImmRmediated inhibitory mechanism, which suppresses integration of incoming ICEBs1 element into the recipient chromosome, is circumvented in MICE system as it only transfers a plasmid and need not to be integrated into the recipient chromosome. The ConG-YddJ-mediated exclusion mechanism remains to be overcome in both mini-ICEBs1 and the MICE systems. Nevertheless, our MICE-mediated conjugation resulted high efficiency in various B. subtilis strains, similar to that of the mini-ICEBs1 system reported earlier (Brophy et al., 2018). Thus, MICE-mediated conjugation efficiency is sufficiently high to transfer plasmids into ICEBs1-containing recipient strains, although the ConG-YddJ-mediated exclusion mechanism may reduce the efficiency.
This study showed that natural competence could not transfer plasmids to undomesticated B. subtilis strains tested (Figure 1). The result may be due to a lack of natural competence-related genes or the negative interplay between biofilm formation and competence (She et al., 2020). Some strains may have competence inhibitor genes (Konkol et al., 2013). This study also showed that the plasmid was introduced into four out of eight undomesticated B. subtilis strains via electroporation (Figure 1). The method showed inconsistent results, indicating that optimization of the electroporation conditions for each strain is required (Figure 1). Several conjugation methods, such as RP4 transfer system (Simon et al., 1983), pLS20 plasmid system (Koehler and Thorne, 1987), and ICEBs1 (Johnson and Grossman, 2015) have been reported in Bacillus strains. In this study, the EBC using the RP4 transfer system, and the BBC using the pLS20 plasmid showed consistent results and broader range compared to the natural competence and electroporation methods, but some undomesticated Bacillus strains still remained untransformed (Figure 1). The newly developed MICE system in this study showed a wider host range than other systems. It transformed not only undomesticated B. subtilis but also QPS Bacillus species (Figures 3, 4). The reason for the wide host range can possibly be the increased availability of conjugation machinery due to the overexpression a regulator RapI that induces the expression of the MICE genes. The applicability of MICE system in a broad-host-range shall guide the future studies on the marker-free genome editing of other microorganisms beyond Bacillus species.
CRISPR-Cas9-mediated genome engineering has been well established with high efficiency in B. subtilis (Altenbuchner, 2016;Westbrook et al., 2016;So et al., 2017). Since the CRISPR-Cas9 (B) SDS-PAGE analysis of the total protein extracted from the 24-h culture supernatants of the QPS Bacillus strains. Protein molecular mass markers are indicated on the "M" lanes. The lanes marked with "W," "E," and "A" represent wild type, edited, and aprN-expressing strains, respectively. The size of AprN mature protease was 27.7 kDa, indicated by arrows. system requires a gRNA and two flanking DNA fragments for homologous recombination, plasmids for simultaneous multigenome editing are not simple in construction and large in size, which can reduce transformation efficiency. Moreover, simultaneous multiple editing with Cas9 produces multiple fragmentation of the chromosome, which can result in very low editing efficiency. There is a recent report of simultaneous editing of three targets for point mutations using Cas9 nickase in B. subtilis (Liu et al., 2019). It showed considerable mutation efficiency, but still needed donor DNA fragments. A recently developed CBE system enables precise base editing in yeast, mammalian cells, and bacteria without double-stranded break (Komor et al., 2017). The independence of donor DNA fragments can simplify the cloning process and reduce the size of the plasmid. A CBE system for B. subtilis has been reported as a powerful tool for multiplex genome editing due to its characteristics of simplicity, high efficiency, high specificity, and low genomic damage. It has generated offtarget mutations in about 8.5 single nucleotide variants in CBE-edited strains, but most off-target mutations can be silent or harmless (Kim et al., 2021). Even if there are detrimental mutations that affect major cellular physiology, they can be filtered out by subsequent screening of growth and physiological characteristics. In this study, we showed that the CBE system can simultaneously edit multiple genes in various Bacillus species as well as B. subtilis, indicating that various undomesticated Bacillus strains isolated from environments could be engineered for cell factories. In the future, various other CRISPR tools could be transferred into undomesticated Bacillus strains using the MICE system for cell factory engineering.
Bacillus strains have been isolated from various environments, such as soil, plant, food, marine sediments, and the human gut with a wide range of pH, temperatures, and salts. They produce versatile enzymes and secondary metabolites and are notable resources for the discovery of novel antibiotics (Aleti et al., 2015;Zhao and Kuipers, 2016;Grubbs et al., 2017). In addition to traditional fields, such as industry and agriculture, Bacillus strains are considered to have great potential in medical applications. To realize their potential, Bacillus research should be shifted from laboratory strains to undomesticated ones. Therefore, the systems for engineering undomesticated strains developed in this study could allow for breakthroughs in research for the utilization of untapped Bacillus resources.


Data on body mass, glucose tolerance and bone phenotype of mice with osteogenesis imperfecta on long-term low-fat and high-fat diets

Male and female mice with a dominant severe bone fragility disorder, osteogenesis imperfecta, and their wild-type littermates (FVB background) were challenged with a long-term (26 weeks) high-fat diet to evaluate the development of obesity and glucose intolerance. Here we present data for the measurements of body mass, the outcome of glucose tolerance tests during the long-term diet, as well as organ weights and bone phenotype at the end of the study. Interpretation of the data and further in-depth analysis can be found in the article “Male but not female mice with severe osteogenesis imperfecta are partially protected from high-fat diet-induced obesity.” by Tauer JT, Boraschi-Diaz I, Al Rifai O, Rauch F, Ferron M, Komarova SV, published in Molecular Genetics and Metabolism. The data presented here demonstrate individual mouse outcomes of long-term diet experiments that can be reused for comparative studies of diet-induced changes in wild-type mice on different backgrounds and different mouse models of osteogenesis imperfecta.

induced changes in wild-type mice on different backgrounds and different mouse models of osteogenesis imperfecta.  Table   Subject Endocrinology, Diabetes and Metabolism Specific subject area Long-term high-fat diet in a mouse model with dominant severe osteogenesis imperfecta. Type of data Graph, Figure  How

Value of the Data
• The presented data are useful for researchers interested in long-term high-fat diet-induced metabolic response in a mouse model of osteogenesis imperfecta and their FVB wild-type littermates. • The presented data are beneficial for researchers investigating the influence of diet-induced obesity on bone phenotype in mice. • The presented data are valuable to understand the relationship of diet-induced obesity on glucose metabolism and osseous phenotype in relation to sex in mice. • The data presented might be reused for comparative studies of diet-induced changes in wildtype mice on different backgrounds and in different mouse models of osteogenesis imperfecta.

Data Description
Interpretation of the data and further in-depth analysis can be found here [1] . All raw individual mouse outcomes of long-term diet experiments can be found in the excel file available by the Mendeley database [2] . The excel file contains individual mouse data about body mass development (excel sheet 1), performed glucose tolerance tests (GTT) (excel sheet 2), calculated GTT-related area under the curve (excel sheet 3), fasted glucose levels (excel sheet 4), absolute organ weights (excel sheet 5), and femoral structural (excel sheet 6) and biomechanical parameters (excel sheet 7).
In brief, data describe long-term diet-induced weight gain in WT and OI mice and its possible effects on glucose homeostasis and bone properties. Fig. 1 depicts glucose homeostasis measurements during 26-week diet-intervention in male and female WT and OI mice. Fig. 2 depicts absolute organ weights of liver, pancreas, lung, heart, spleen, brown adipose tissue (BAT), and white adipose tissue (WAT) in male and female WT and OI mice at the end of the study. Fig. 3 depicts regression analysis of fasted glucose levels during long-term diet regimen in male and female WT and OI mice. Fig. 4 depicts selected femoral structural and mechanical properties in male and female WT and OI mice at the end of the study. Further, femoral structural and mechanical parameters can be found here [1] and in the Mendeley database.

Fig. 2. Absolute organ weights in WT and OI mice after long-term low-fat (LFD) or high-fat (HFD) diet . Data represent mean
± standard error of mean (SEM). Animal numbers were as follows: male WT LFD n = 6; male WT HFD n = 9; female WT LFD n = 7; female WT HFD n = 10; male OI LFD n = 7; male OI HFD n = 13; female OI LFD n = 2; female OI HFD n = 9. Statistical analysis: two-way ANOVA with Bonferroni post-test. Significances below 0.99 are indicated by either p-value or significance symbol as * p < 0.05, * * p < 0.01, * * * p < 0.001. Further, significances are color coded and demonstrate diet effect: black, and genotype effect: blue. §, Statistical analysis might be compromised by low animal number of this group.

Animal experiment, treatment, and data collection
Experimental design and treatment are described here [1] . In brief, the Col1a1 Jrt/ + mouse on FVB background was used as mouse model for dominant moderate-to-severe Osteogenesis imperfecta (OI) [ 4 , 5 ]. Col1a1 Jrt/ + mice carry a splice site mutation in exon 9 of the COL1A1 gene leading to an 18-amino acid deletion in the collagen type I α1 chain [4] . Col1a1 Jrt/ + mice are smaller in size and demonstrate OI bone phenotype with shorter long bones, lower bone volume/tissue volume (BV/TV), and development of spontaneous fractures [4] . The breeding colony was maintained at the Animal Care Facility of the Shriners Hospitals for Children-Canada. Animals were on a 12 h alternating light and dark cycle and had unrestricted access to water and food. Prior to long-term diet regimen, mice were fed a regular chow (Rodent chow #5075, Charles River Laboratories). Male and female wild-type (WT) and OI mice were challenged with either a high-fat/low-sugar diet (HFD; Teklad Custom Diet, #TD.06414) or control low-fat/lowsugar diet (LFD; Teklad Custom Diet, #TD.180127), starting at an age of 4 weeks until 30 weeks of age (diet length: 26 weeks). Diets were purchased from Envigo (Huntingdon, UK) and were supplemented with vitamin mix containing vitamin D 3 and vitamin K 1 . Detailed diet composition can be found in the Supplement of the related research article [1] . Diets were stored at 4 °C before usage. Food was replaced weekly. Individual body mass was recorded weekly using a portable compact balance (OHAUS Corporation, Model CS200, Parsippany, US). Individual glucose homeostasis was evaluated by glucose tolerance tests every 4-weeks. After 26-weeks of diet, mice were anesthetized using isoflurane inhalation and terminated by intracardiac puncture followed by cervical dislocation. Heart, lung, liver, spleen, interscapular brown adipose tissue (BAT), inguinal white adipose tissue (WAT), and pancreas were isolated, weighted using analytical balance (Denver Instrument GmbH, Model P-114, Goettingen, Germany), and snap-frozen in a 1.5 mL Eppendorf tube using liquid nitrogen and stored at −80 °C. Right and left femora were isolated, length measured with a digital caliper (Starrett, Model EC799A-6/150, Athol, USA), and stored in a 1.5 mL Eppendorf tube at −20 °C in phosphate buffered saline-soaked gauze until further analysis.

Glucose tolerance test (GTT) during long-term high-fat diet
GTT was performed as described before [1] . Food was withdrawn for 5 h for 4-week-old mice, or for 16 h for older mice, prior to GTT. Blood glucose was measured using single-use glucose strips with the ONETOUCH VerioFlex glucometer (LifeScan Europe, Zug, Switzerland; maximum measurement limit 33.3 mmol/L) in samples from tail tip immediately before (time 0) and 15, 30, 60, 90, and 120 min after intraperitoneal injection of sterile 2 g/kg glucose solution (glucose dissolved in tris-buffered saline, pH 7.4). Area under the curve (AUC) was calculated by using the trapezoid rule [6] .

Ex vivo micro-computed tomography
Micro-computed tomography's of the right femora were performed using Skyscan 1272 (Bruker, MA, US). Prior to micro-computed tomography measurements, femora were thrown at 4 °C overnight. Following day, wrapped femora were taken out of the 1.5 mL Eppendorf tube and placed vertically into a 0.5 mL Eppendorf tube with the proximal end facing upwards and the distal end downwards. Afterwards, 0.5 mL Eppendorf tube was placed 'up-side-down' on the respective scanning holder (final femoral scanning position: distal bone end facing upwards and proximal bone end facing downwards). The entire femur was scanned, applying the following scanning parameters a voxel size of 5 μm, a 0.40-degree increment angle, 3 frames averaged, a 66 kV and 142 mA X-ray source with a 0.5 mm Al filter to reduce beam-hardening artefacts. Scans were reconstructed using Skyscan NRecon software (Bruker Optics Inc., Billerica, MA, USA) with correction of X/Y alignment, and identical beam hardening and ring artifact corrections applied to all samples. Afterwards, reconstructed scans were rotated using Skyscan Dataviewer Software (Bruker Optics Inc., Billerica, MA, USA) to ensure 100% vertical positioning of the femora prior to analysis. Trabecular bone was analyzed in reconstructed and rotated scans in a region starting at 0.5 mm proximal of the distal femoral growth plate (to avoid primary spongiosa) and scanning a section spanning 10% of the femoral length in a proximal direction. Trabecular bone was automatically segmented from the inner cortical surface and quantified using the system's analysis software (Skyscan CT Analyser, Version 1.16.1.0). To analyze cortical bone, analysis was performed at the middle of the femur spanning a section of 5% of the femoral length. The software derives outer bone diameter and the diameter of the bone marrow cavity from cross-sectional areas using a circular bone cross-section model. Cortical thickness was calculated as the difference of these two diameters divided by 2. Scanned hydroxyapatitemimicking phantoms (0.25 and 0.75 g/cm 3 Ca-HA) were used to allow for bone mineral density (BMD) calculation. Phantoms were scanned by applying the parameters mentioned above  : (A, B) non-linear regression analysis of LFD-fed and HFD-fed mice with the same genotype and evaluation of statistical differences between curve slopes. (C, D) Non-linear regression analysis of WT and OI mice on either LFD or and HFD with evaluation of statistical differences between intercept and slope of the curves. Star symbol: statistical outliers, detected by ROUT method [3] , not included in regression analysis.

Fig. 4. Femoral structural (A-D) and biomechanical parameters (E-G) after long-term diets in male and female WT and OI
mice . Data represent mean ± standard error of mean (SEM). LFD, low-fat diet; HFD, high-fat diet; Tb.Sp., trabecular separation; trab. BMD, trabecular bone mineral density. Statistical analysis: three-way ANOVA with Bonferroni post-test. Significances below 0.99 are indicated by either p-value or significance symbol as * p < 0.05, * * p < 0.01, * * * p < 0.001. Further, significances are color coded and demonstrate diet effect: black, genotype effect: blue, sex effect: red. and BMD calculations were performed according to Bruker method note "Bone mineral density (BMD) and tissue mineral density (TMD) calibration and measurement by micro-CT using Bruker-MicroCT CT-Analyser".

Three-point-bending test
Following ex vivo micro-computed tomography scanning, right femora were loaded to failure in three-point bending using a Materials Testing System Model 5943 (INSTRON, Norwood, MA, USA). The bones were cleaned off of all muscle tissues and soaked overnight in 1X phosphatebuffered saline (PBS) at room temperature until mechanical testing. Individual femur was placed horizontally on two lower supports with the distal end facing to the left and the proximal end facing to the right side. The distance between the lower supports was 7 mm. The anterior middiaphysis was loaded under tension via a third support with a vertical displacement rate of 50 μm/s. Test results were recorded using the system's analysis software Bluehill (Illinois Tool Works Inc., Glenview, IL, USA; Version 3.65). Femoral stress and elastic modulus were calculated as described before [7] .

Statistics
Unless stated otherwise, data presented in figures are shown as mean ± standard error of mean (SEM). Statistical differences were assessed using GraphPad Prism version 7.0d (GraphPad Software, San Diego, California, USA). Either two-way ANOVA testing or three-way ANOVA testing with Bonferroni post-test were applied. P < 0.05 was considered for all tests significant. For non-linear regression analysis, outliers, detected by ROUT method [3] , were excluded from the calculation and are displayed in the figures as stars.

Ethics Statement
All experiments were approved by the Animal Care Committee of McGill University (Montreal, Canada; protocol number: 2012-7127), complied with ARRIVE guidelines, and conformed to the ethical guidelines of the Canadian Council on Animal Care. Male and female WT and Col1a1 Jrt/ + mice were used in this study. Male WT and female WT and OI mice demonstrated diet-induced weight gain during long-term HFD diet, while male OI mice are protected from HFD-induced weight gain [1] . HFD-fed male OI mice did not show any diet effects on organ weights, while female OI mice demonstrated increased WAT weights on HFD. Fragile OI bone phenotype persisted on LFD and HFD. Evaluation of sex-related differences between male and female mice on LFD and HFD demonstrated higher mechanical properties in female WT than male WT mice, while female OI mice showed only higher stiffness than male OI mice.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships which have or could be perceived to have influenced the work reported in this article.


Copper and nanostructured anatase rutile and carbon coatings induce adaptive antibiotic resistance

Contaminated surfaces are vehicles for the spread of infectious disease-causing microorganisms. A strategy to prevent their spread is applying antimicrobial coatings to surfaces. Both nanostructured anatase rutile and carbon (NsARC), a TiO2 formulation, and copper are examples of antimicrobial agents that are used in making or coating door handles and similar surfaces, to reduce microbial loads. Antimicrobial surfaces have been extensively tested for antimicrobial activity but not sublethal effects, such as exposure-associated multiple antibiotic resistance phenotypes usually caused by induction of efflux pump genes. The possibility of NsARC and copper inducing indicative efflux pump pathways was investigated by monitoring the expression of mScarlet fluorescent protein (FP) in two reporter strains of Escherichia coli. There was an increase in the expression of FP in the reporter strains exposed to NsARC and copper relative to the inert control composed of stainless steel. Furthermore we tested E. coli and Staphylococcus aureus following 8 h of exposure to NsARC for changes in resistance to selected antibiotics. E. coli that were exposed to NsARC became more susceptible to kanamycin but there was no significant change in susceptibility of S. aureus to any tested antibiotics. These findings suggests that even though NsARC and copper are antimicrobial, they also have some potential to cause unintended phenotypes. Supplementary Information The online version contains supplementary material available at 10.1186/s13568-022-01457-z.

Introduction
Nanostructured anatase rutile and carbon (NsARC), a composite of titania and carbon is an antimicrobial surface coating that is active with or without photoactivation (Wasa et al. 2021). The killing mechanism of NsARC has not been precisely described but at least involves free radicals that are released sequel to photoexcitation of TiO 2 (Krumdieck et al. 2019), active carbon-centred free radicals (Fenoglio et al. 2009) and desiccation of cells over time (Yu et al. 2014). NsARC with antimicrobial properties could be of value because disease-causing microorganisms are becoming resistant to other available methods of prevention and treatment (Djurišić et al. Open Access *Correspondence: jack.heinemann@canterbury.ac.nz 2015; Leung et al. 2016). Prescription medicines, disinfectants, and other antimicrobial agents are not uniformly effective against disease-causing microorganisms and the use of any can select for reduced susceptibility to the others (Dancer 2008;Foster et al. 2011;Kampf 2018). Therefore, the diseases caused by these microorganisms are becoming more difficult, sometimes impossible, to treat with antibiotics (Heinemann 1999;Metcalfe et al. 2016;Williamson et al. 2015).
Exposure of microorganisms to toxic metals is unavoidable and desirable. Toxic metals are sometimes found in high concentrations in the environment as a result of natural geological events such as volcanos and or human activities such as mining, smelting, fossil fuel burning and other industrial activities (Ali et al. 2019;Barkay et al. 2010). Microorganisms have evolved mechanisms by which they can both acquire and maintain essential metals at physiologically relevant concentrations, and eliminate them when they are in excess (Ali et al. 2019;Chandrangsu et al. 2017). They have a variety of strategies for limiting toxicity such as extracellular and or intracellular sequestration of metals, modification of target sites, reduction in outer membrane porins (OMP), enzymatic detoxification and or increase in efflux of metals (Nies 2003), and biofilm formation. Resistance to nanomaterials such as NsARC might be more difficult for microorganisms to evolve. Nanomaterials can have multiple cellular targets and therefore the options available for microorganisms to mitigate the effects of nanomaterials is limited (Lemire et al. 2013;Wright et al. 2006).
Nevertheless, sub-lethal exposures might still result in unintended effects as has been observed for other antimicrobial agents. Exposing bacteria to non-antibiotic chemicals can predispose them to develop resistance to antibiotics (Jun et al. 2019;Kurenbach et al. 2015Kurenbach et al. , 2017Kurenbach et al. , 2018. Salmonella enterica serovar Typhimurium exposed to sublethal concentration of the herbicides Kamba ® and 2,4-D grew at higher concentrations of ampicillin, chloramphenicol, ciprofloxacin and tetracycline antibiotics (Kurenbach et al. 2015). Escherichia coli exposed to the fungicide copper ammonium acetate also grew on medium with higher concentrations of tetracycline and E. coli exposed to atrazine grew on higher concentrations of ciprofloxacin, kanamycin and streptomycin (Jun et al. 2019). Food preservatives and emulsifiers used in food and medicines could also cause these types of changes (Kurenbach et al. 2017;Molina-González et al. 2014). The way bacteria respond after exposure to various chemicals may vary depending on their genetic and physiological differences (Chiang and Schellhorn 2012).
Should NsARC be used on a commercial scale for its antimicrobial properties, we wanted to know if there were unintended outcomes that we could anticipate. We previously hypothesized that sub-lethal exposures to NsARC could cause similar changes to antibiotic susceptibility (Wasa et al. 2021). This is based on the innate response of bacteria to toxic environments. The innate response includes but is not limited to changes in expression of efflux and permeability. Common markers for this response are tolC and soxS genes (Alderton et al. 2021). Changes in the expression of these two genes can alter susceptibility pattern of E. coli to antibiotics. This was investigated comparing NsARC, copper and stainless steel.

Bacterial strains, culture conditions, materials and chemicals
Bacterial strains used in this study are shown in Table 1. E. coli BW25113 (Baba et al. 2006) is a useful strain for genetic studies because it the parent in a series that includes nearly every possible gene knockout. We have measured the baseline responses of efflux phenotypes and gene expression using this strain (Kurenbach et al. 2017). Strains are stored in 15% (v/v) glycerol solution at − 80 °C. They were recovered for use on Lauria Bertani (LB) agar plates (Lennox-L-Broth Base, Invitrogen, Auckland (New Zealand) and agar (Bacteriological Agar No.1, Oxoid, Hampshire (UK)) and then incubated at 37 °C. Plates were replaced at one-week intervals. All antibiotics were purchased from Sigma, Auckland (New Zealand). Paraformaldehyde used for fixation of cells prior to fluorescence microscopy was purchased from Sigma, Auckland (New Zealand) and stored as powdered stock at 4 °C. 4% (v/v) solution of paraformaldehyde was made up on the day it was to be used and kept at 4 °C.
Stainless steel (25 × 25 mm) coupons were used as an inert control material as recommended by the ISO 27447:2009 (Mills et al. 2012), and commercially pure copper (25 × 25 mm) coupons were selected as a positive control material because they have known antimicrobial properties that are independent of light (Wasa et al. 2021). The NsARC (25 × 25 mm) coupons and control (stainless steel and copper) samples were sterilised by immersion in 70% ethanol and the NsARC test pieces were then aseptically stored in the dark for > 48 h. Gelatine coated slides were made by immersing glass microscopy slides (Mareinfeld-Superior, 76 × 26 mm, approx. 1 mm thick) in 70% ethanol for 1 h, then air drying and dipped in 0.1% (v/v) gelatine solution at 70 o C before air drying again. After drying the slides were kept at 4 °C and used within 1 week of drying.

Determining if NsARC can cause a change in susceptibility of E. coli and S. aureus to antibiotics
The E. coli culture was exposed to the tests and control metal samples using a modified version of ISO 27447:2009 (Wasa et al. 2021). 100 µl of the test organisms containing ~ 1,000,000 cells at the stationary growth phase were placed on 25 × 25 mm NsARC and stainlesssteel surfaces and 24 × 24 mm sterile coverslip (Lab Supply, Dunedin (New Zealand)) was place on top, causing the liquid to spread evenly on the surface. The samples were then placed in 60 × 15 mm petri dishes. Replicates were then simultaneously exposed to visible light of 2100 lx (450-650 nm) or kept in the dark for 8 h. Afterwards, the samples were rinsed using phosphate buffered saline (PBS). The number of bacteria in 100 µl of the PBS was about ~ 10 2 cells. This volume was transferred to the surface of LB plates containing 0, 0.01, 0.02, 0.03, 0.04, 0.05 µg/ml of ciprofloxacin, or 0, 5, 6, 7, 8, 9 µg/ml of chloramphenicol, or 0, 4, 5, 6, 8, 9 µg/ml of kanamycin or 0, 0.5, 0.7, 1, 1.5, 2 µg/ml of tetracycline.
The same procedure as followed using Staphylococcus aureus. Wash off from NsARC was transferred to the surface of LB plates containing 0, 1, 2, 3, 4, 5, 6 µg/ml of erythromycin, or 0, 0.09, 0.1, 0.2, 0.3, 0.4 µg/ml of fusidic acid, or 0, 0.9, 1, 2, 3, 4 µg/ml of kanamycin, or 0, 0.07, 0.08, 0.09, 0.1, 0.2 µg/ml of oxacillin, or 0, 0.09, 0.1, 0.2, 0.3, 0.4 µg/ml of tetracycline or 0, 0.5, 0.8, 1, 1.5, 2 µg/ml of vancomycin. The E. coli and S. aureus wash off from stainless steel was diluted threefold to achieve ~ 10 2 cells and then was transferred to the surface of LB plates containing various concentration of antibiotics as described above. The minimum inhibitory concentrations of the antibiotics are shown in Additional file 1: Table S21. Plates were incubated at 37 °C for 24 h. All experiments were conducted three times to obtain biological replicates. Three samples of each (test and control) were used for each experiment to obtain technical replicates. The plates were monitored for up to three days. This was compared for the various treatments (material and exposure conditions). There were variations in the values, thus, the cfu/ml counts were normalised to efficiency of plating (EOP) values using the formula.
The EOP values were then used to plot graphs using graphpad prism software.

Determining if NsARC can induce gene expression
Individual colonies formed by reporter strains E. coli BWtolC and BWsoxS (Jun et al. 2019) were placed into LB broth (Lennox-L-Broth Base, Invitrogen, Auckland (New Zealand)) supplemented with kanamycin and then placed on a shaker platform at 37 °C and grown to exponential phase. 100 µl containing about ~ 10 7 cells was then placed on separate NsARC (test), stainless steel (negative control) and copper (positive control) coupons. Sterile cover slips (24 mm × 24 mm) were used to spread the cultures on the sample surfaces. The samples were placed in petri dishes (60 mm × 15 mm) containing damp filter paper. Replicates were simultaneously exposed to visible light of 2100 lx (450-650 nm), UV light (365 nm), ambient light (650-750 nm) and also kept in the dark for a period of not more than 2 h before washing off with PBS and the cells fixed with paraformaldehyde (Chao and Zhang 2011). 2 µl of the fixed cells were then smeared onto a gelatine-coated glass slide and allowed to dry at room temperature. 6 µl of 50% (v/v) glycerol was then added to the dried smear and a 25 × 25 mm glass cover slip was used to spread it over the dried smear.

Microscopy and image processing
The fixed cells were examined with an Axio Imager.M1 (Zeiss, Oberkochen, Germany) using 556/20 nm excitation bandpass. Digital images were captured at 100⋅ magnification with an AxioCam MRm camera (Zeiss) in phase contrast and through a 556/20 nm (red) filter set.
Approximately 10 NsARC and 10 control samples each were used for this experiment. And about 50 images were captured from each individual sample. A total of 500 images from each treatment was taken for each treatment replicate. Images were then analysed using Fiji ImageJ (Schindelin et al. 2012). 100 separate images for each sample were analysed. Single-cell fluorescence intensity of the individual cells was obtained by acquiring multichannel images of the fluorescence signals and phase contrast signals. Multichannel images were imported into the Fiji ImageJ program. Thresholding command and standard settings were used to separate cells from the background based on the phase contrast. The analyse particles command was then used to add cells to the region of interest manager and the average fluorescence of the individual cells was determined using the "multimeasure" command (Remus-Emsermann et al. 2016).
The relative fluorescence is also just an arbitrary unit (au) of measurement.

Statistical analysis
R was used for statistical analysis (Rosario-Martinez et al. 2015). We used analysis of variance (ANOVA) to analyse the data from the experiment we carried out to determine if exposure of bacteria to NsARC caused a change in susceptibility to antibiotics. A multifactor ANOVA was performed on EOP values to test for effect of the materials (NsARC and control) and antibiotic concentration under two exposure conditions (light and dark). Residual plots were examined to determine if EOP values were normally distributed, which is an assumption for ANOVA (Crawley 2007). The plots were not normally distributed. So, the EOP scores were log transformed to meet the assumption.
In each case, we tested for significant difference between materials. The null hypothesis was that there was no difference between the EOP values from the materials at various antibiotic concentrations. We also tested for interaction between materials, antibiotic concentrations and exposure conditions. A Bonferroni's post hoc test was used to compare the EOP to determine if there is a significant difference between NsARC and the controls. The value for statistical significance was set at P < 0.05. The results of each post hoc are available in supplementary material (Additional file 1: Table S1-S12), however, we were most interested in the differences in EOP between individual treatment combinations as follows: NsARC vs. steel under light and NsARC vs. steel in the dark. Contrast matrices listing the contrast of interest mentioned were drawn up and the test Interactions function in the phia package in R was used to evaluate the contrasts as described in the result section (Rosario-Martinez et al. 2015).
For the experiment to determine if NsARC can induce changes in the expression pattern of genes that can alter susceptibility to antibiotics, a statistical model based on ANOVA was also used. A Tukey's post hoc test was used to compare the means of the relative fluorescence to determine if there is a difference between NsARC and the controls. The value for statistical significance was set at P < 0.05. The results of each post hoc are available in supplementary material (Additional file 1: Table S13-S20). However, we were most interested in the differences between individual treatment combinations, these include NsARC vs. Positive control (copper), Negative control (stainless steel) vs. Positive control (copper) and NsARC vs. Negative control (stainless steel). These were calculated using contrasts and the results can also be found in the supplementary materials (Additional file 1: Table S13-S20). Violin plots were then made using ggplot2 (Wickham 2016).

Change in susceptibility to antibiotics
10 2 cells of E. coli and S. aureus that had survived exposure to NsARC with and without light for about 8 h were tested for responses to selected antibiotics. E. coli were challenged with different concentrations of ciprofloxacin, chloramphenicol, kanamycin, and tetracycline and S. aureus with different concentrations of kanamycin, vancomycin, erythromycin, oxacillin, tetracycline and fusidic acid. Changes in response to each antibiotic because of the previous exposure to NsARC were shown as a differential efficiency of plating (EOP) (Figs. 1, 2). No significant change in susceptibility to tetracycline (Additional file 1: Table S3), chloramphenicol (Additional file 1: Table S4) and ciprofloxacin (Additional file 1: Table S5) was observed (Additional file 1: Fig. S1). A significant change (P < 0.001) in the EOP was observed for E. coli exposed to both light and NsARC or only NsARC (Fig. 1). The significant change was in the direction of lowering the minimum inhibitory concentration of kanamycin after exposure to NsARC.
No significant change in susceptibility to any antibiotic was observed for S. aureus that had survived exposure to NsARC with or without light ( Fig. 2; Additional file 1: Fig.  S2).

tolC and soxS can be induced by NsARC
Two reporter strains each expressing either PtolC-mScarlet or PsoxS-mScarlet were designed and constructed as described previously (Jun et al. 2019). These two strains of E. coli "report" changes in transcription of the tolC and soxS genes. We and others have found that these genes are often induced in response to stress (Fernández and Hancock 2012), particularly oxidative stress (Demple 1996).
Each of these reporter strains had a plasmid construct that expressed an mScarlet red fluorescent protein under the control of the promoters of either the tolC or soxS genes. These designed constructs are strains wherein an increase in expression of fluorescent protein is expected when the tolC or soxS genes are induced (Jun et al. 2019). After exposure to NsARC, stainless steel and copper, the single cell fluorescence intensities were measured.
Overall, the PtolC − mScarlet reporter strain was brighter than the reporter strain expressing PsoxS − mScarlet (Fig. 3). There were also variations in the fluorescence of both reporters when exposed to either NsARC, stainless steel or copper regardless of light conditions (high intensity visible, UV, ambient light and no light) (P < 0.001) indicating that both material type and exposure conditions contribute to the variation in fluorescence intensities. Both reporter strains were brighter on copper under all exposure conditions compared to when they were on either NsARC or stainless steel (Figs. 4,5). The reporter strain expressing PtolC − mScarlet and exposed to UV light (Fig. 4A) was significantly brighter on copper than when exposed to UV and NsARC (P < 0.001) or UV and stainless steel (P < 0.001). The bacteria that were on NsARC were significantly brighter (P < 0.001) than the ones that were on stainless steel. Under high-intensity visible light (Fig. 4B), reporter strains that were on NsARC were significantly brighter (P < 0.001) than the ones on stainless steel. However, under ambient light (Fig. 4C), reporter strains on NsARC were not significantly brighter (P = 0.357) than on steel. In the dark (Fig. 4D) reporter strains on NsARC were not significantly brighter (P = 0.479) than the ones on stainless steel.
Under all light exposures PsoxS-mScarlet reporter strains were brighter on copper than on either NsARC of stainless steel. Combined UV light and NsARC exposures (Fig. 5A) caused greater fluorescence than combined UV light and stainless steel (P < 0.001). High-intensity visible light and NsARC caused greater fluorescence (P = 0.003) than combined UV light and stainless steel (Fig. 5B). Fluorescence for combined ambient light and NsARC exposures was also greater than (P = 0.010) combined ambient light and stainless steel exposures (Fig. 5C). Finally, in the dark (Fig. 5D), reporter strains were significantly brighter (P = 0.009) on NsARC than on stainless steel.

Discussion
E. coli and S. aureus exposed to combinations of nonantibiotic antimicrobials and antibiotics demonstrate altered responses to antibiotics (Alderton et al. 2021). The responses can result in bacteria that survive at antibiotic concentrations above the previous minimal inhibitory concentration, have a survival or growth advantage at sub-lethal concentrations, or the opposite in that they die at lower concentrations or have a growth disadvantage at lower concentrations of antibiotic. In all cases the frequency of evolution of clinically relevant resistance increases when the bacteria are exposed to antibiotics at concentrations that alter their fitness (Alderton et al. 2021;Kurenbach et al. 2018).
It is unknown if exposures to coating materials have a similar effect on microbes. We tested this using E. coli and S. aureus that have survived exposure to NsARC. We observed that there was a significant increase in susceptibility of E. coli to one antibiotic, kanamycin, with no significant change in susceptibility to the other antibiotics tested. There was also no change in the susceptibility of S. aureus to any of the antibiotics that were tested after exposure to NsARC.
We also used fluorescent reporter strains to determine if tolC and soxS genes were transcriptionally induced by exposures to copper or NsARC. Reporters that were exposed to copper were significantly brighter than when exposed to NsARC or stainless steel. The increase in fluorescence of the reporter strains that were exposed to copper is not surprising because we have earlier reported a similar increase in fluorescence when the reporter strain expressing PtolC − mScarlet was cultured in media supplemented with copper (Jun et al. 2019). Copper is a known inducer of soxS and tolC transcription (Franke et al. 2003;  Nishino et al. 2007), and can also induce marR, making marR an agent that acts as a copper sensor (Hao et al. 2014). The increase in cell fluorescence may be due to oxidative activity of the copper (Fenoglio et al. 2009).
Reporter strains that were exposed to NsARC and UV or high intensity light were significantly brighter than the strains that were on stainless under the same conditions. Fluorescence levels were the same for bacteria that were exposed to either NsARC or stainless steel and either ambient light or no light. The major reason for the difference in brightness in the presence or absence of light is not known, but possibly it is because light excitation of NsARC is needed in order to facilitate the production of ROS and other free radicals that can induce soxS and tolC genes (Koutsolioutsou et al. 2005;Verdier et al. 2014). Absence of light excitation affects the production of free radicals and hence these gene are not induced with no corresponding effect on the brightness of the cell.
Aside from the potential usefulness of NsARC for its antimicrobial properties, there was a need to also consider the possible potential adverse effects NsARC may have. NsARC is a biocidal material, and like other antimicrobial agents, there is a possibility that it may induce higher levels of antibiotic resistance. The fact that the reporter strains that were on NsARC were brighter than the ones on stainless steel indicated that NsARC may be able to induce genes that also have effects on antibiotic toxicities. From this study, we could not explain the mechanism by which NsARC was inducing the genes because we only tested a specific efflux pump component and a regulatory protein.
We also could not quantify how much of this increase in fluorescence accounts for the increase in the expression of efflux pumps. Whole transcriptome sequencing could provide an overview of mRNA levels of most genes that might be affected by NsARC.
Additional file 1: Fig. S1.Response of E. coli to various concentrations of (A) Tetracycline (C)Chloramphenicol (E) Ciprofloxacin after surviving exposure to stainless steeland NsARC in the dark and (B) Tetracycline (D) Chloramphenicol (F)Ciprofloxacin after surviving exposure to stainless steel and NsARC undervisible light.